As part of the current laser-like focus on the diagnosis and tracking of infectious disease, several classes of molecules have been targeted as potential biomarkers and therapeutic targets, including microRNAs (miRNAs). MiRNAs are emerging as key mediators of the host response to infection via the regulation of genes involved in innate and adaptive immunity (Drury, O’Connor, & Pollard, 2017). They have also been shown to be stable in biofluids, control the cellular tropism of certain viruses, have a role in individual resistance to some infections like HIV, and are associated with impaired vaccine response in older people. Recent evidence is also implicating them in the response to SARS-CoV-2/COVID-19. Recognition of this pivotal role has led to their increasing prominence in infectious disease research and drug development.

In this article, we will outline the benefits and recent advancements of miRNA in the field of infectious disease.

Current diagnostic limitations in infectious disease

Infectious diseases currently account for approximately 15% of total global mortality (Organization & 2016, n.d.). Due to the most recent global pandemic as well as the ease of travel in our current society, there is a critical need to diagnose infections early and to assist in containing spread of disease. Biomarkers allow us to predict, diagnose, control, and monitor disease. Right now, there is a pressing need for biomarkers that allow for early diagnosis in situations where control is important or currently difficult.

The present landscape for detection of infectious disease is relatively diverse and involves identification of the pathogen itself or the host’s immune response to the pathogen. Identification becomes more complex when an unknown agent with an unknown response arises because current identification methods, including protein assays and molecular diagnostics such as PCR, require substantial time to establish and can suffer from technical limitations. Among the questions that arise are ones about false positives and false negatives due to low sensitivity, low specificity, or poor sample collection.

Without an understanding of immune response, antibody testing provides low value in monitoring pandemic spread. For example, certain viruses such as rabies (Ito, Moseley, & Sugiyama, 2016) and cytomegalovirus (Patro, 2019) efficiently evade or alter the immune response. Additional complications for certain disease also make early detection impossible or rely solely on post-mortem tissue for diagnosis.

As we look retrospectively, it is clear that advancements in diagnostic biomarkers are needed to circumvent these issues.

miRNAs as biomarkers

miRNAs first gained traction outside the infectious disease field when miR-15 and miR-16 were identified as tumor suppressors for chronic lymphocytic leukemia (Calin et al., 2002). Evidence that they played a role in the immune system first arose from mouse studies showing that production of mature B and T lymphocytes is correlated to miRNA expression (Drury et al., 2017).

MiRNAs regulate and reflect a number of biological processes via regulation of gene expression. As many pathologies and phenotypes are a result of dysregulated gene expression, tracking mRNA levels and the means by which they are regulated helps to understand underlying mechanisms. Thousands of these molecules have been identified and many have been mapped to cellular processes that are key to healthy function such as development, homeostais, neurobiology, immunology, and response to infections. For an in-depth review, please see Saliminejad, Khorram Khorshid, Soleymani Fard, & Ghaffari, 2019.

MiRNAs are present in many biofluids such as blood, urine, and saliva, which allows for non-invasive, quick, and versatile sample collection. They are also highly stable in these fluids and thus easier to process and work with. Not only that, but recent work has shown that miRNAs that originate in one part of the body also circulate throughout the body. Both immune and non-immune cells show release of miRNAs into the extracellular environment (Robbins et al., 2012). The basis for miRNAs as biomarker signatures is linked to their precise expression at different pathological states.

Use of miRNAs as biomarkers of infectious disease

Currently, miRNAs have been profiled as potential biomarkers in: Hendra virus (Cowled et al., 2017), HIV (Biswas, Haleyurgirisetty, Lee, Hewlett, & Devadas, 2019), tuberculosis (Lyu et al., 2019), malaria (Li et al., 2018), and Ebola (Duy et al., 2016). In addition, circulating serum miRNAs have been used to distinguish latent and active tuberculosis, to generate early prognostic signatures of sepsis, and as biomarkers of disease severity in viral hepatitis caused by hepatitis B and C virus (“MicroRNAs in Infectious Disease: Emerging Clinical Applications—Infectious Disease Advisor,” n.d.). In some cases, such as the study by Biswas et al., miRNAs were observed earlier than disease onset, even before the pathogen could be detected, and prior to the onset of seroconversion.

While self-limiting conditions or those that require minimal intervention such as the common cold may not benefit from development of miRNA platforms, others, specifically those with a large global impact, current lack of diagnostic tools, or long incubation periods would benefit the most from such technology. The potential of miRNAs in disease makes a myriad of improvements conceivable including early detection, pathogen identification, detection of latent infections, measurement of treatment efficacy, prediction of disease outcomes and host response, and guiding optimal therapeutic selection as well as companion diagnostics. For more details refer to Tribolet et al., 2020.

miRNAs in respiratory disease

MiRNAs have also been implicated in a number of respiratory disorders including influenza infections (Scheller et al., 2019) and rhinoviruses (Hasegawa et al., 2018).

One important biological function of miRNAs is the maintenance of the epithelial cell barrier in the respiratory tract and regulation of antiviral response (Głobińska, Pawełczyk, & Kowalski, 2014). Since viral acute respiratory infections (ARIs) are the main cause of acute respiratory symptoms (e.g., flu, bronchitis, and SARS-CoV-2), the expression profile of miRNAs in epithelial cells may contribute to the pathogenesis of these acute respiratory disease. Therefore, the potential to improve diagnostic capabilities for other respiratory viruses, such as the recent severe acute respiratory syndrome (SARS-CoV-2) outbreak, is not to be understated.

miRNAs as signatures in SARS-CoV-2

Although much of the research on SARS-CoV-2 is preliminary, an analysis of current literature and bioinformatic approaches that examined potential human miRNA interactions with the SARS-CoV-2’s genome and compared the miRNA target sites to seven other coronaviruses showed that pathogenic human coronaviruses had miRNA signatures that differ from non-pathogenic human coronaviruses. A set of unique miRNAs was identified for COVID-19 while SARS and MERS had their own unique signatures. The research team proposed that pathogenic coronaviruses could modulate host miRNA levels by acting as “miRNA sponges to facilitate viral replication and/or to avoid immune responses” (Bartoszewski et al., 2020).

Additional studies, which point in the same direction, include the following: Arisan et al., 2020; Guterres, de Azeredo Lima, Miranda, & Gadelha, 2020; Hosseini Rad SM & McLellan, 2020. Along the same lines, there is evidence that the increased virulence of COVID-19 in the aging population could be explained by a decreased abundance of miRNAs (Fulzele et al., 2020).

Research involving miRNAs as potential therapeutic targets in SARS-CoV-2 is focused on the angiotensin-converting enzyme (ACE2), which has been reported as a COVID-19 entry receptor that regulates host cell infection (Lu et al., 2020). Work is underway to see if miRNA molecules that target ACE2 can be used to regulate the SARS-CoV-2 receptor in specific tissue types that are susceptible to disease pathology (Chauhan et al., 2020).

Obstacles

The lack of specificity among miRNAs responding to diverse infections is a major hurdle to effective use of miRNAs as biomarkers in infectious disease. The need for more robust methods for identifying and evaluating predictive miRNA signatures is also an issue, although machine learning has already been used to identify miRNA biomarkers and will likely increase. Additionally, standards need to become more common place for quantification and detection of miRNA levels.

Emerging miRNA platforms

Current methods for miRNA detection include northern blotting, microarrays, mass spectrometry, qPCR, and next-generation sequencing with the latter two being much more common due to higher sensitivity and specificity. More recent technologies aim to improve on detection and quantification of miRNA focusing on making methods that are portable, reliable, robust, rapid, specific, sensitive, low-cost, and user-friendly to facilitate the implementation of miRNAs in a clinical setting. If miRNAs are going to transition from lab-based research to point-of-care technologies, these improvements are particularly essential. Great attention has been paid to tactics that can increase efficiency of miRNA extraction or to implement robust detection methods such as digital microfluidic devices and electrochemical biosensor. For a comprehensive overview of the ways that research is aiming to transform miRNA detection platforms into point-of-care devices, please see Tribolet et al., 2020.

Conclusion

With the world in the middle of a global pandemic, it is clear that development of novel biomarker technologies is desperately needed. MiRNAs have the potential to serve as biomarkers of infection with bacteria, parasites, viruses, and even prions. Given time, the limitations of miRNAs in the lab can be overcome and they can serve as a powerful new tool in both diagnostics and therapeutics for infectious disease.

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